GSM6589894: P. syringae-treated Arabidopsis leaf (bulk, replicate 3); Arabidopsis thaliana; RNA-Seq
Protoplasts were isolated from Arabidopsis leaves infiltrated by bacteria and 10 mM MgCl 2 (mock) using the Tape-Arabidopsis Sandwich method. Peeled leaves were immediately immersed in a petri dish containing 10 mL of enzyme solution (1.5% Cellulase Onuzuka R-10 (Yakult, Japan), 0.3% Macerozyme R-10, (Yakult, Japan), 0.4 M Mannitol, 20 mM KCl, 20 mM MES (2-(N-Morpholino)ethanesulfonic acid hydrate) pH 5.7, 10 mM CaCl2, and 0.1%, BSA). After digesting for 100 min with gentle shaking, the protoplast suspension was filtered through 40 μm cell strainer (BD Falcon 352340) into a round-bottomed 50 mL tube and centrifuged at 100 x g for 1 min at 22°C using a swinging rotor. Protoplast pellets were gently resuspended in 10 mL of CS-sucrose buffer (0.4 M sucrose, 20 mM MES pH 5.7, 20 mM KCl) and centrifuged at 100 x g for 2 min at 22°C. Intact and healthy protoplasts remained suspended in the upper layer. The upper layer suspension was then transferred into a clean round-bottomed tube and gently mixed with 10 mL of protoplast buffer (0.4 M Mannitol, 20 mM KCl, 20 mM MES pH 5.7, and 0.1%, BSA). After centrifuging at 100 x g for 2 min at 4°C using a swinging rotor, the supernatant was removed without disturbing the loosely packed protoplast pellets. Leaf samples were processed cDNA libraries were prepared with NEBNext Multiplex Oligos for Illumina sequencing